The Israeli scorpion Leiurus quinquestriatus hebraeus produces a venom which is known to inhibit a number of different potassium (K.sup.+) channel pathways in vertebrates. These include two different classes of calcium (Ca.sup.2+)-activated K.sup.+ channels that are inhibited by different venom components: an apamin-sensitive Ca.sup.2+ -activated K.sup.+ channel, such as that found in guinea pig hepatocytes; and an apamin-insensitive Ca.sup.2+ -activated K.sup.+ channel, such as found, for example, in human erythrocytes, Ehrlich cells and rat thymocytes (Abia, A., et al., Biochem. Biophys. Acta., 856, 403 (1986), Castle, N. A. , and Strong, P. N., FEBS Lett. 209, 117 (1986))
Of the many constituents present in the scorpion's crude venom, one minor component is a toxin (called charybdotoxin) that has been shown to block reversibly a Ca.sup.2+ -activated K.sup.+ channel isolated from rat skeletal muscle and reconstituted into planar lipid bilayers (Miller et al., Nature, 313, 316 (1985)). Charybdotoxin (ChTX), appears to be a small molecular weight basic protein that only inhibits channel activity when added at the external face of the channel protein. An initial report on ChTX confirmed this protein to be a high affinity selective inhibitor of the high conductance (ca. 200 picosemens; 200 pS) Ca.sup.2+ -activated K.sup.+ channel found in the plasma membrane of many vertebrate cells (Smith, C., et al , J. Biol. Chem. 261, 14607 (1986)).
Smith et al. in J. Biol Chem. also included a characterization of the physical and chemical properties of the toxin, wherein ChTX was reported to have an apparent molecular weight of approximately 10 KDa, to be unusually stable to organic solvents or heat treatment, and to inhibit Ca.sup.2+ - activated K.sup.+ channel function with an apparent dissociation constant of 3.5 nM. Subsequently, ChTX was also found to inhibit low conductance (ca. 35 pS) Ca.sup.2+ -activated K.sup.+ channels in neurons from the marine mollusk Aplysia californica, but not block sodium (Na.sup.+), Ca.sup.2+, transient K.sup.+ or delayed rectifying K.sup.+ channels in this preparation (Herman, A., and Erxleben, C., J. Gen. Physiol. 90, 27, (1987)).
ChTX, therefore, represents the only described agent known to cause potent specific inhibition of the apamin insensitive class of Ca.sup.2+ -activated K.sup.+ channels. However, to be a useful probe for these channels, ChTX must be purified to homogeneity from the crude venom, which contains many different activities, and its structure must be correctly elucidated.
The present invention discloses for the first time the purification of ChTX to homogeneity, the chemical structure of this peptide, and the biological activity of the pure toxin, which had been previously mischaracterized by Smith et al. in terms of its purity, molecular weight, amino acid composition and N-terminal amino acid residue.